Synthesis of 2-alkyl amino acids

ABSTRACT

Non-natural amino acids such as 2-alkylated amino acids allow for the synthesis of a wider variety of peptidal and non-peptidal pharmaceutically active agents. A method of preparing a 2-alkyl amino acid involves a Michael-type addition of a nucleophile to a dialkyl 2-methylidenylpropan-1,3-dioate and the conversion of a ester moiety into an amino moiety. The present invention also discloses a method of preparing a class of iron chelating agents related to desferrithiocin, all of which contain a thiazoline ring. In this method, an aryl nitrile or imidate is condensed with cysteine, a 2-alkyl cysteine, or a cysteine ester.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/438,770 (now allowed) filed May 15, 2003 now U.S. Pat. No.7,002,036. U.S. application Ser. No. 10/438,770 claims the benefit ofU.S. Provisional Application Nos. 60/381,012, 60/381,021, 60/380,894,60/380,910, 60/380,880, 60/381,017, 60/380,895, 60/380,903, 60/381,013,60/380,878 and 60/380,909, all of which were filed May 15, 2002. U.S.application Ser. No. 10/438,770 also claims the benefit of U.S.Provisional Application No. 60/392,833, filed Jun. 27, 2002. The entireteachings of the above-referenced applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Alpha-amino acids are useful starting materials in the synthesis ofpeptides, as well as non-peptidal, pharmaceutically activepeptidomimetic agents. In order to enable the synthesis of a largenumber of compounds from an amino acid precursor, it is advantageous tohave naturally occurring and non-naturally occurring amino acids.Non-naturally occurring amino acids typically differ from natural aminoacids by their stereochemistry (e.g., enantiomers), by the addition ofalkyl groups or other functionalities, or both. At this time, theenantiomers of naturally occurring amino acids are much more expensivethan the naturally occurring amino acids. In addition, there are only alimited number of commercially available amino acids that arefunctionalized or alkylated at the alpha-carbon, and often synthesesinvolve the use of pyrophoric or otherwise hazardous reagents. Moreover,the syntheses are often difficult to scale up to a commercially usefulquantity. Consequently, there is a need for new methodologies ofproducing such non-naturally occurring amino acids.

Non-naturally occurring amino acids of interest include the (R)- and(S)-isomers of 2-methylcysteine, which are used in the design ofpharmaceutically active moieties. Several natural products derived fromthese isomers have been discovered in the past few years. These naturalproducts include desferrithiocin, from Streptomyces antibioticus; aswell as tantazole A, mirabazole C, and thiangazole, all from blue-greenalgae. These compounds have diverse biological activities ranging fromiron chelation to murine solid tumor-selective cytotoxicity toinhibition of HIV-1 infection.

Desferrithiocin, deferiprone, and related compounds represent an advancein iron chelation therapy for subjects suffering from iron overloaddiseases. Present therapeutic agents such as desferroxamine requireparenteral administration and have a very short half-life in the body,so that patient compliance and treatment cost are serious problems forsubjects receiving long-term chelation therapy. Desferrithiocin andrelated compounds are effective when orally administered, therebyreducing patient compliance issues. Unfortunately, (S)-2-methylcysteine,which is a precursor to the more active forms of desferrithiocin andrelated compounds, remains a synthetic challenge. Therefore, there is aneed for novel methods of producing 2-methylcysteine at a reasonablecost, and means of isolating the desired enantiomer.

SUMMARY OF THE INVENTION

The present invention includes a method of preparing a compoundrepresented by Structural Formula (I):

or a salt thereof;wherein:

-   -   R₁ is —H or a substituted or unsubstituted alkyl group;    -   R₂ is a substituted or unsubstituted alkyl group; and    -   R₃ is —H, —SH, —OH, —NH₂, —CO₂H, —CONH₂, —NHC(NH)NH₂, a        substituted or unsubstituted alkyl group, a substituted or        unsubstituted cycloaliphatic group, a substituted or        unsubstituted heterocyclic group, a substituted or unsubstituted        aromatic group, or a substituted or unsubstituted heteroaromatic        group, wherein R₃ optionally comprises a protecting group;        comprising the steps of:    -   a.) reacting a nucleophile of the formula A—R₃ or A—(R₃)₂, with        a compound represented by Structural Formula (II):

-   -   -   wherein:        -   A is —H, —Li, —CuLi, —MgCl, —MgBr, or —MgI, provided that A            and R₃ are not each —H;        -   R₄ is —H or a substituted or unsubstituted alkyl group; and        -   R₁ and R₃ are as defined above;        -   thereby forming a compound represented by Structural Formula            (III):

-   -   b.) reacting the product of step (a.) with one or more bases,        R₂X, and a phase transfer catalyst,        -   wherein X is a leaving group; and        -   R₁, R₂, R₃, and R₄ are as defined above;        -   thereby forming a compound represented by Structural Formula            (IV):

-   -   c.) converting the product of step (b.) into a compound        represented by Structural Formula (V):

-   -   d.) optionally cleaving the protecting group of R₃, thereby        forming the compound represented by Structural Formula (I).

In another embodiment, the present invention is a method of preparing acompound represented by Structural Formula (VII):

or a salt thereof;wherein:

-   -   R₆ is —H or a substituted or unsubstituted alkyl group; and    -   R₇ is a substituted or unsubstituted alkyl group        comprising the steps of:    -   a.) reacting a nucleophile, A—S—Z, with a compound represented        by Structural Formula (VIII):

-   -   -   wherein:        -   A is —H;        -   R₈ is —H or a substituted or unsubstituted alkyl group;        -   Z is a protecting group; and        -   R₆ is as defined above;        -   thereby forming a compound represented by Structural Formula            (IX):

-   -   b.) reacting the product of step (a.) with one or more bases,        R₇X, and a phase transfer catalyst,        -   wherein X is a leaving group; and R₆, R₇, R₈, and Z are as            defined above;        -   thereby forming a compound represented by Structural Formula            (X):

-   -   c.) converting the product of step (b.) into a compound        represented by Structural Formula (XI):

-   -   d.) removing Z from the product of step (c.), thereby forming        the compound represented by Structural Formula (VII).

Preferably, R₁ and R₆ are methyl and R₄ and R₈ are t-butyl.

The above methods can additionally comprise the step of resolvingenantiomers or diastereomers of a 2-alkyl amino acid (or an ester or asalt thereof). Preferably, the method comprises isolating the (R)- and(S)-enantiomers of 2-alkyl amino acids, or esters or salts thereof.

The present invention also includes a method of preparing a compoundrepresented by Structural Formula (XVI):

comprising the step of coupling (S)-2-methylcysteine or a salt thereof,as prepared by a method described herein, to 2,4-dihydroxybenzonitrile.Alternatively, an analogous compound can be synthesized by coupling2-hydroxybenzonitrile and (S)-2-methylcysteine or a salt or an esterthereof. Similar syntheses can be conducted with other substitutedbenzonitriles.

Advantages of the present invention include the facile synthesis of a2-alkyl amino acid from a dialkyl 2-methylidenylpropan-1,3-dioate.Additional advantages include the ability to prepare amino acids with awide variety of side chains, such as preparing 2-methylcysteine.2-Methylcysteine prepared by the method of the present invention can becoupled to 2,4-dihydroxybenzonitrile to form4′-hydroxydesazadesferrithiocin, also referred to as4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid, an iron chelating agent.

DETAILED DESCRIPTION OF THE INVENTION

A useful and efficient method of preparing 2-alkyl amino acids involvesa Michael-type addition of a side chain precursor to a dialkyl2-methylidenylpropan-1,3-dioate, followed by alkylation at the2-position of the diester. One of the ester groups can be converted intoan amino moiety, typically through a reaction with an azide.

Michael-type additions of the present reaction include reacting anucleophile of the formula A—R₃ or A—(R₃)₂ with a dialkyl2-methylidenylpropan-1,3-dioate, which forms a 2-substitutedpropan-1,3-dioate ester. Typically, when the nucleophile does notcontain a metal-carbon bond, the Michael-type addition occurs in aprotic solvent with either a catalytic amount of a base or astoichiometric amount of base. When the nucleophile contains ametal-carbon bond such as a lithium-carbon, copper-carbon, or magnesiumcarbon, the Michael-type addition occurs under conditions where thenucleophile is stable and adds to the dialkyl2-methylidenylpropan-1,3-dioate at the desired location. Reactiontemperature is generally not important, however, the temperature canrange from −50° C. to 150° C., 0° C. to 100° C., or 20° C. to 60° C.Michael additions are further described on pages 741-742 and 797-803 of“Advanced Organic Chemistry, Fourth Edition,” by Jerry March,Wiley-Interscience, 1992 and references therein, all of which areincorporated by reference.

Preferred nucleophiles include nucleophiles where R₃ is —SH, such as H₂Sand CH₃COSH. Other suitable examples of R₃ include —H,—(CH₂)_(x)S(CH₂)_(y)H, —(CH₂)_(x)O(CH₂)_(y)H, —(CH₂)_(x)NH(CH₂)_(y)H,—(CH₂)_(x)C(O)NH₂, —(CH₂)_(x)C(O)OH, —(CH₂)_(x)NHC(NH)NH₂, a C1-C6substituted or unsubstituted alkyl group, and aryl and heteroaryl groupssuch as

where R₃ optionally comprises a protecting group. The variable x can bean integer of zero or more, such as 0 to about 6, preferably 0-3, or 0or 1. The variable y is 0 or 1.

Alkylation of a 2-substituted propan-1,3-dioate ester (forming a2-alkyl-2-substituted propan-1,3-dioate ester) typically occurs in aprotic solvent (e.g., methanol, ethanol, water, propanol, isopropanol,formic acid, acetic acid, DMF, N-ethylacetamide, formaldehyde diethylacetal), by adding one or more bases, an alkylating agent of the formulaR₂X or R₇X, and a phase transfer catalyst. Suitable bases include alkalimetal or alkaline earth metal hydroxides, alkoxides, or carbonates suchas sodium hydroxide, potassium hydroxide, sodium methoxide, potassiummethoxide, sodium ethoxide, potassium ethoxide, sodium carbonate, cesiumcarbonate, calcium carbonate and potassium carbonate, as well as sodiumhexamethyl disilazide and potassium hexamethyl disalizide. Preferredalkylating agents include those where R₂ or R₇ is a C1-C4 substituted orunsubstituted alkyl group and X is a halide. Especially preferredalkylating agents include those where R₂ or R₇ is methyl or benzyl and Xis a halide such as iodide. Examples of phase transfer catalysts includebenzyl triethyl ammonium chloride, benzyl trimethyl ammonium chloride,benzyl tributyl ammonium chloride, tetrabutyl ammonium bromide,tetraethyl ammonium bromide, tetrabutyl ammonium hydrogen sulfate,tetramethyl ammonium iodide, tetramethyl ammonium chloride,triethylbutyl ammonium bromide, tributyl ethyl ammonium bromide,tributyl methyl ammonium chloride, 2-chloroethylamine chloride HCl,bis(2-chloroethyl)amine HCl, 2-dimethylaminoethyl chloride HCl,2-ethylaminoethyl chloride HCl, 3-dimethylaminopropyl chloride HCl,methylamine HCl, dimethylamine HCl, trimethylamine HCl, monoethylamineHCl, diethylamine HCl, triethylamine HCl, ethanolamine HCl,diethanolamine HCl, triethanolamine HCl, cyclohexylamine HCl,dicyclohexylamine HCl, cyclohexylamine HCl, diisopropylethylamine HCl,ethylenediamine HCl, aniline HCl, methyl salicylate, ethyl salicylate,butyl salicylate amyl salicylate, isoamyl salicylate, 2-ethylsalicylate,and benzyl salicylate.

Converting a 2-alkyl-2-substituted propan-1,3-dioate ester into a2-alkyl amino acid typically comprises a series of steps where the2-alkyl-2-substituted propan-1,3-dioate ester is partially hydrolyzed(at R₄ and R₈) to give a free carboxylic acid, optionally converting thefree carboxylic acid into an acid chloride, and the free carboxylic acidor the acid chloride is reacted with a source of azide and water to givethe amino acid. Typically, hydrolysis is achieved by treating the2-alkyl-2-substituted propan-1,3-dioate ester with acid. This hydrolysisis additionally applicable to other esters. The optional conversion intoan acid chloride can be accomplished by reacting the free carboxylicacid with an agent such as SOCl₂, PCl₃, or ClC(O)C(O)Cl. The source ofazide for an acid chloride is MN₃, where M is H or an alkali metal. Thesource of azide for a free carboxylic acid is preferablydiphenylphosphoryl azide. Following reaction with a source of azide, acarboxy azide is formed, and further reaction with water and heatresults in the carboxy azide rearranging into isocyanate. The isocyanatecan readily be hydrolyzed to an amino moiety.

Alternatively, the above conversion can involve amidating a2-alkyl-2-substituted propan-1,3-dioate ester. Typically, amidationinvolves hydrolyzing the ester to a free carboxylic acid, converting thefree carboxylic acid to an acid chloride (or directly converting theester to an acid chloride), and reacting the acid chloride with ammoniaor a salt thereof. The amide can be converted to an amino moiety byreacting it with 1.) MOR₅ and Y₂ or 2.) MOY; where M is an alkali metal,R₅ is hydrogen, or an alkyl group such as methyl, ethyl, propyl, orisopropyl; and Y is a halogen.

If R₃ comprises a protecting group, it can be cleaved. Similarly, aprotecting group Z can be cleaved. Cleavage of a protecting group isdependent on the nature of the protecting group. For example, an acylprotecting group can be removed by treating the protecting group withacids such as hydrochloric acid, acetic acid, dilute sulfuric acid, andthe like; and bases such as sodium hydroxide, potassium hydroxide,sodium methoxide, potassium methoxide, sodium ethoxide, and potassiumethoxide. Other examples of removing protecting groups can be found in“Protective Groups in Organic Synthesis, 3^(rd) Edition” by Peter G. M.Wuts and Theodora W. Greene, Wiley-Interscience, 1999, which isincorporated herein by reference.

R₁, R₆, and R₉ can optionally be hydrolyzed from the amino acid product.Typically, hydrolysis is achieved by reacting the ester form of an aminoacid with a sufficient quantity of acid or base to remove R₁, R₆, or R₉.The acid or base used for hydrolysis preferably does not react with orcleave, except to form a salt, other moieties of the amino acid.

Amino acid products, either enantiomers or diastereomers, of the abovesyntheses can be resolved. Typically, amino acids are resolved byforming a diastereomeric salt with an amino acid and a chiral amine.Suitable chiral amines include arylalkylamines such as(R)-1-phenylethylamine, (S)-1-phenylethylamine, (R)-1-tolylethylamine,(S)-1-tolylethylamine, (R)-1-phenylpropylamine, (S)-1-propylamine,(R)-1-tolylpropylamine, and (S)-1-tolylpropylamine. Resolution of chiralcompounds using diastereomeric salts is further described in CRCHandbook of Optical Resolutions via Diastereomeric Salt Formation byDavid Kozma (CRC Press, 2001), incorporated herein by reference in itsentirety.

Alternatively, 2-alkyl amino acids and functionalized derivativesthereof (e.g., esters) can be resolved by emulsion crystallization, asdescribed in U.S. Pat. Nos. 5,872,259, 6,383,233 and 6,428,583, whichare incorporated herein by reference. Briefly, emulsion crystallizationis a process for separating a desired substance from an aggregatemixture. The process involves forming a three phase system, the firstphase comprising the aggregate mixture, the second phase being liquidand comprising a transport phase, and the third phase comprising asurface upon which the desired substance can crystallize. A chemicalpotential exists for crystal growth of the desired substance in thethird phase of the system, thereby creating a flow of the desiredsubstance from the first phase through the second phase to the thirdphase, where the desired substance crystallizes and whereby anequilibrium of the activities of the remaining substances in theaggregate mixture is maintained between the first phase and the secondphase.

In one example of emulsion crystallization, a solution of the racemicmixture is supersaturated (by either cooling, adding a solvent in whichone or more components are sparingly soluble or by evaporation of thesolution). Ultrasonication typically aids the process of forming anemulsion. The mixture is then seeded with crystals of the desired,optically active acid along with an additional quantity of surfactantand an anti-foaming agent. The desired product usually crystallizes outand can be separated by filtration. Further details of emulsioncrystallization for an amino acid derivative can be found in Example 3.

Once the 2-alkyl amino acids or functionalized derivatives have beenresolved, the desired isomer can be isolated. Typically, a (S)-2-aminoacid, a salt, or an ester thereof is isolated. Preferably,(S)-2-methylcysteine or (S)-2-methylcysteine methyl ester is isolated.

Cysteine, a 2-alkylcysteine such as (S)-2-methylcysteine, or a cysteinealkyl ester can be coupled to a substituted or unsubstituted arylnitrile such as a substituted or unsubstituted benzonitrile. Preferably,the substituents on benzonitrile will not interfere with the couplingreaction. In a preferred embodiment, (S)-2-methylcysteine is coupled to2,4-dihydroxybenzonitrile to form4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as 4′-hydroxydesazadesferrithiocin).

Typically, coupling of cysteine, a 2-alkylcysteine, or a cysteine alkylester and a substituted or unsubstituted benzonitrile includesconverting the benzonitrile into a benzimidate. The benzimidate can beformed, for example, by reacting the benzonitrile with an alcohol suchas methanol, ethanol, n-propanol, or isopropanol in the presence of anacid such as hydrochloric acid. Alternatively, cysteine or a relatedcompound can be coupled directly with a benzimidate. The benzimidate isthen reacted with the cysteine (or related compound) under basicconditions. Acceptable bases include trimethylamine, triethylamine,triphenylamine, dimethylamine, diethylamine, diphenylamine,diisopropylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane(DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and the like. Thereaction between the benzimidate and the cysteine results in thethiazoline (4,5-dihydrothiazole) containing product. When forming thebenzimidate from a hydroxylated benzonitrile (e.g.,2,4-dihydroxybenzonitrile), the hydroxyl groups are advantageouslyprotected (e.g., with a substituted or unsubstituted alkyl or arylalkylgroup such as a benzyl group). The protecting groups are subsequentlycleaved, typically by catalytic hydrogenation.

The methods of the claimed invention can be used to manufacture otherrelated desferrithiocin analogs and derivatives. Examples of suchanalogs include those described in U.S. Pat. Nos. 5,840,739, 6,083,966,6,159,983, 6,521,652 and 6,525,080 to Raymond J. Bergeron, Jr., thecontents of which are incorporated herein by reference. Additionalexamples can be found in PCT/US93/10936, PCT/US97/04666, andPCT/US99/19691, the contents of which are incorporated by reference.

Suitable benzonitriles and benzimidates for use in the above couplingreaction can be synthesized by methods described in U.S. ApplicationNos. 60/381,013, 60/380,878 and 60/380,909, all filed May 15, 2002, theentire teachings of which are incorporated herein by reference.

An alkyl group is a hydrocarbon in a molecule that is bonded to oneother group in the molecule through a single covalent bond from one ofits carbon atoms. Alkyl groups can be cyclic or acyclic, branched orunbranched, and saturated or unsaturated. Typically, an alkyl group hasone to about 24 carbons atoms, or one to about 12 carbon atoms. Loweralkyl groups have one to four carbon atoms and include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl.

A cycloaliphatic group is cyclic, non-aromatic, consists solely ofcarbon and hydrogen and may optionally contain one or more units ofunsaturation, e.g., double and/or triple bonds. A cycloaliphatic groupcan have one or more rings, which can be fused together. Typically, acycloaliphatic group has one to about 24 carbons atoms, or about 1 toabout 12 carbon atoms. Examples of cycloaliphatic groups includecyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl,cyclopentenyl, cyclopenta-1,3-dienyl, cyclohexenyl,cyclohexa-1,3-dienyl, cyclohexa-1,4-dienyl, cycloheptenyl, cyclooctenyl,cycloocta-1,3-dienyl, and cycloocta-1,3,5-trienyl.

A heterocyclic group is a cycloaliphatic group where one or more of thecarbon atoms is replaced by a heteroatom such as S, O, or N. Examples ofheterocyclic groups include oxiryl, oxetyl, oxolyl, oxyl, aziridyl,azetidyl, pyrrolidyl, piperidyl, tetrahydrothiophyl, andtetrahydrothiopyryl.

Aromatic (or aryl) groups include carbocyclic aromatic groups such asphenyl, p-tolyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.Heteroaromatic groups include N-imidazolyl, 2-imidazole, 2-thienyl,3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl,4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic, alicyclic, or aromatic ring or heteroaryl ring isfused to one or more other heteroaryl or aryl rings. Examples include2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl,2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazole,2-benzooxazole, 2-benzimidazole, 2-quinolinyl, 3-quinolinyl,1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl.

Suitable substituents for alkyl and cycloaliphatic groups include —OH,halogen (—Br, —Cl, —I and —F), —O(R′), —O—CO—(R′), —CN, —NO₂, —COOH, ═O,—NH₂, —NH(R′), —N(R′)₂, —COO(R′), —CONH₂, —CONH(R′), —CON(R′)₂, —SH,—S(R′), and guanidine. Each R′ is independently an alkyl group or anaromatic group. Alkyl and cycloaliphatic groups can additionally besubstituted by a heterocyclic, aromatic, or heteroaromatic group (e.g.an alkyl group can be substituted with an aromatic group to form anarylalkyl group). A substituted alkyl or cycloaliphatic group can havemore than one substituent.

Suitable substituents for heterocyclic, aromatic, and heteroaromaticgroups include —OH, halogen (—Br, —Cl, —I and —F), —O(R′), —O—CO—(R′),—CN, —NO₂, —COOH, ═O, —NH₂, —NH(R′), —N(R′)₂, —COO(R′), —CONH₂,—CONH(R′), ′CON(R′)₂, —SH, —S(R′), and guanidine. Each R′ isindependently an alkyl group or an aromatic group. Heterocyclic,aromatic, and heteroaromatic groups can additionally be substituted byan alkyl or cycloaliphatic group (e.g. an aryl group can be substitutedwith an alkyl group to form an alkylaryl group such as tolyl). Asubstituted heterocyclic, aromatic, or heteroaromatic group can havemore than one substituent.

Functional groups of the present invention can be protected with aprotecting group. For example, cysteine or a related compound isprotected at one or more reactive moieties, such as at the amino, —SH,and/or carboxyl moieties of cysteine. As is known in the art, aprotecting group reduces or eliminates the ability of a functional groupto react with another functional group. For example, a thiol or analcohol can be protected with an acyl group. Similarly, an alcohol canbe protected by a tosyl or a trimethylsilyl group. An amine can, forexample, be protected by an Fmoc group or a Boc group. Additionalprotecting groups, methods of adding a protecting group, and methods ofremoving a protecting group are taught in “Protective Groups in OrganicSynthesis, 3^(rd) Edition” by Peter G. M. Wuts and Theodora W. Greene,Wiley-Interscience, 1999, which was incorporated by reference above.

Protecting groups for basic nitrogen atoms include formyl;4-toluenesulfonyl; t-butyloxycarbonyl; 2,4-dinitrophenol;benzyloxymethyl; trityl; t-butoxymethyl; 2-chlorobenzyloxy-carbonyl;allyloxycarbonyl; benzyloxycarbonyl (Z); mesitylene-2-sulfonyl;4-methyloxy-2,3,6-trimethyl-benzyenesulfonyl;2,2,5,7,8-pentamethyl-chroma n-6-sulfonyl; 9-xanthenyl; and2,4,6-trimethoxybenzyl.

Protecting groups for basic sulfur groups include 4-methylbenzyl,3-nitro-2-pyridinesulfenyl; trityl; 2,4,6-trimethoxybenzyl;acetamidomethyl; trimethylacetaminomethyl; t-butylsulfonyl; andsulfoxide.

Protecting groups for basic oxide groups include benzyl ether; t-butylether; benzyl ether; 2,6-dichlorobenzyl ether; 2-bromobenzyl ether; and3,5-dibromobenzyl ether.

Carboxyl groups can be protected, for example, as ethers or ascarboxamides. For example, when a carboxyl group is protected as anether, it takes the form of —COOR wherein R is a substituted orunsubstituted C1 to C10 alkyl group, a substituted or unsubstituted upto C30 alkyl group, or a substituted or unsubstituted alkyl-aryl groupwherein the alkyl group is C1 to C5 and the aryl group is up to C30.When a carboxyl group is protected as a carboxamide, it takes the formof —CONR′ wherein R′ is —H or as in R above.

Leaving groups are typically weak bases. Suitable leaving groups includehalogen, tosyl, triflyl, brosyl, p-nitrophenyl, 2,4-dinitrophenyl, andmesyl groups. Halogens include bromine, chlorine, and iodine.

Also included in the present invention are salts of the disclosed aminoacids and amino acid esters (including side chains). For example, aminoacids can also be present in the anionic, or conjugate base, form, incombination with a cation. Suitable cations include alkali metal ions,such as sodium and potassium ions, alkaline earth ions, such as calciumand magnesium ions, and unsubstituted and substituted (primary,secondary, tertiary and quaternary) ammonium ions. Suitable cations alsoinclude transition metal ions such as manganese, copper, nickel, iron,cobalt, and zinc. Basic groups such as amines can also be protonatedwith a counter anion, such as hydroxide, halogens (chloride, bromide,and iodide), acetate, formate, citrate, ascorbate, sulfate or phosphate.

EXAMPLE 1

A one-necked, 100 mL, round-bottomed flask was fitted with Dean-Starkapparatus attached with a drying tube (CaCl₂) and a magnetic stirrer.The flask was charged with 5 g (26.6 mmol) of t-butyl ethyl malonate,2.39 g (78.8 mmol) of formalin solution (35% formaldehyde in water), 3.4g (40 mmol) of piperidine and 50 mL of toluene. The mixture was heatedto reflux with stirring in an oil bath at 120-130° C. for 8 hours. Aftercooling to room temperature, toluene was removed under reduced pressure.The crude oily product was purified by column chromotography on silicagel, and was eluted with ethyl acetate/petroleum ether (6:94) to give3.56 g (67%) of t-butyl ethyl methylene malonate.

¹H NMR (CDCl₃, 200 MHz) δ 1.35 (t, 3H), 1.55 (s, 9H), 4.30 (q, 2H), 6.40(d, 2H).

To a one necked 50 mL round-bottomed flask fitted with a refluxcondenser and an outlet dipped inside aqueous KMnO₄ solution, 1.0 g (5mmol) of t-butyl ethyl methylenemalonate and 1.4 g thiol acetic acid(18.4 mmol) was heated under reflux for 12 hours. The mixture wasallowed to cool, and the product was purified by silica gel columnchromatography using ethyl acetate/petroleum ether (7:93) to afford 0.86g (62%) of t-butyl ethyl acetylthiomethylmalonate as a colorless liquid.

¹H NMR (CDCl₃, 200 MHz) δ 1.35 (t, 3H), 1.50 (s, 9H), 2.35 (m, 3H), 2.52(m, 2H), 3.30 (m, 1H), 4.20 (m, 2H).

EXAMPLE 2

Methyl t-butyl 2-methylidenyl-1,3-dipropionate is reacted withthioacetic acid to form methyl t-butyl2-acetylthiomethyl-1,3-dipropionate. Methyl t-butyl2-acetylthiomethyl-1,3-dipropionate is alkylated with potassiumcarbonate and methyl iodide in the presence of a phase transfer catalystto form methyl t-butyl 2-acetylthiomethyl-2-methyl-1,3-dipropionate. Thet-butyl group is hydrolyzed by acidifying the reaction mixture. The freecarboxylic acid group produced by hydrolyzing the t-butyl group isconverted to an amino group through a reaction with diphenylphosphorylazide, thereby forming S-acetyl-2-methylcysteine methyl ester. Theacetyl group is removed to form 2-methylcysteine methyl ester.

EXAMPLE 3

All compounds were used without further purification. The surfactantsRhodafac RE 610 and Soprophor FL were obtained from Rhône-Poulenc,Surfynol 465 from Air Products, Synperonic NP 10 from ICI and sodiumlauryl sulfate from Fluka. For agitation a shaking machine was used(Buhler KL Tuttlingen). Purities of the resulting crystals were measuredby using a PolarMonitor polarimeter (IBZ Hannover). Ethanol was used asthe solvent. The total crystal quantity was dissolved in a 1 mL cell at20° C.

45 mg of (R,R)- and (S,S)-amino acid derivatives were dissolved in 1 mlof a mixture of 20% v/v 2-hexanol, 12% v/v Rhodafac RE 610, 6% v/vSoprophor FL and 62% v/v water by heating to 80° C. in a 5 mL vial.After the organic derivative was completely dissolved the microemulsionwas cooled down to room temperature and agitated using a shaking machine(420 rpm). During two hours no spontaneous crystallization was observed.The mixture was then seeded with two drops of a dilute, finely groundsuspension of pure (S,S)-(−) amino acid or its ester crystals grownunder similar conditions. After 2 hours of agitation the resultingcrystals were filtered off, washed with water and dried in a gentlenitrogen stream.

EXAMPLE 4

35 mg of R- andS-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4-carboxylic acidwere dissolved in 1 ml of a mixture of 9% N-methyl-pyrrolidone, 9% v/v2-hexanol, 10% v/v Rhodafac RE 610, 5% v/v Soprophor FL and 68% v/vwater by heating to 50° C. in a 5 mL vial. After the product wascompletely dissolved, the microemulsion was cooled down to roomtemperature and agitated with a shaking machine (350 rpm). During twohours, no spontaneous crystallisation was observed. The mixture was thenseeded with two drops of a dilute, finely ground suspension of pureS-product crystals grown under similar conditions. After two hours ofshaking, the resulting crystals were filtered off, washed with water anddried in a gentle nitrogen stream. The procedure yielded 5.4 mg (15.4%)of colorless crystals, with a greater than 90% purity of the Sentantiomer.

EXAMPLE 5

4.00 g (S)-2-methylcysteine hydrochloride (23.3 mmol, 1.0 meq) and 3.14g 2,4-dihydroxy benzonitrile (23.3 mmol, 1.0 meq) were suspended in 40mL ethanol. After degassing this mixture with nitrogen (30 min) 4.95 gtriethylamine (6.8 mL, 48.9 mmol, 2.05 meq) were added. The obtainedsuspension was heated under reflux in an atmosphere of nitrogen for 20hours and then cooled to room temperature. From this suspension ethanolwas evaporated under reduced pressure until an oil (20% of the initialvolume) was obtained. This oil was dissolved in 50 mL water. Thesolution was adjusted to pH 7.5 with 1.20 ml 20% KOH and was extractedtwo times each with 20 mL methyl t-butyl ether (MTBE). The aqueous layerwas separated, adjusted with 20% KOH to pH 11 and again extracted twotimes each with 20 mL MTBE. After separating the aqueous layer the pHwas set with concentrated HCl to 7.5 and traces of MTBE were distilledoff. Then the aqueous solution was acidified with 1.50 ml concentratedHCl to pH 1.5. The product precipitated. This suspension was stirred at4° C. for 1 hour. Then the precipitate was filtered, washed two timeseach with 10 mL water (5° C.) and dried at 45° C. under vacuum. Thereaction yielded 5.17 g (87.6%) of crude4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid product. ¹H-NMR showed no significant impurity.

EXAMPLE 6

2,4-Dibenzyloxybenzonitrile (0.121 mol) was dissolved in 5.85 g (0.127mol) ethanol and 19.4 ml 1,2-dimethoxyethane in a double walled reactor.This solution was cooled to −5° C., stirred and saturated with dry HClgas over 5 hours at 0-3° C. The reaction mixture was stirred overnightat 2-4° C. under nitrogen. During this time, a product crystallized. Thewhite crystals were filtered off, washed with 1,2-dimethoxyethane (5°C., three times each with 13 ml) and dried. A total of 30 of theprotected ethyl benzimidate was isolated (Yield 88.4%, purity 98.9%).

The protected ethyl benzimidate described above was dissolved inmethanol to generate a 10% solution and was catalytically hydrogenatedat room temperature using 5% Pd/C as a catalyst. The reaction wascompleted after 8 hours. The solution was filtered and the solventevaporated to yield the deprotected product as an orange-yellow solid.The reaction yielded 19.6 g (94%) of product.

In contrast, the formation of the imidate with 2,4 dihydroxybenzonitrilewas a low yielding process, generating the desired product in only 20%yield and with less than desired purity.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of preparing a compound represented by Structural Formula (VII):

or a salt thereof wherein: R₆ is —H or a substituted or unsubstituted alkyl group; and R₇ is a substituted or unsubstituted alkyl group; comprising the steps of: a.) reacting a nucleophile, A—S—Z, with a compound represented by Structural Formula (VIII):

wherein: A is —H; R₈ is —H or a substituted or unsubstituted alkyl group; Z is a protecting group; and R₆ is as defined above; thereby forming a compound represented by Structural Formula (IX):

b.) reacting the compound represented by Structural Formula (IX) with one or more bases, R₇X, and a phase transfer catalyst, wherein X is a leaving group; and R₆, R₇, R₈, and Z are as defined above; thereby forming a compound represented by Structural Formula (X):

c.) converting the compound represented by Structural Formula (X) into a compound represented by Structural Formula (XI):

d.) removing Z from the compound represented by Structural Formula (XI), thereby forming the compound represented by Structural Formula (VII).
 2. The method of claim 1, wherein in step (c.), the compound represented by Structural Formula (X) is reacted with a source of azide and water.
 3. The method of claim 2, wherein the source of azide is MN₃ or diphenylphosphoryl azide, wherein M is H or an alkali metal.
 4. The method of claim 3, wherein in Structural Formulas (Vii), (X) and (XI), and R₇X, R₇ is a C1-C4 alkyl group and X in R₇X is a halide.
 5. The method of claim 4, wherein R₇ is methyl.
 6. The method of claim 5, further comprising the step of resolving the enantiomers or diastereomers of the compound represented by Structural Formula (VII).
 7. The method of claim 6, further comprising the step of isolating an (S)-2-amino acid or an ester thereof from the enantiomers or diastereomers of the compound represented by Structural Formula (VII).
 8. The method of claim 7, wherein R₆ is methyl and R₈ is t-butyl.
 9. The method of claim 8, wherein the source of azide is diphenylphosphoryl azide.
 10. The method of claim 9, wherein X is iodide.
 11. The method of claim 10, wherein Z is —C(O)CH₃. 